U.S. patent application number 15/802066 was filed with the patent office on 2018-05-24 for nucleic acid-polymer conjugates for bright fluorescent tags.
The applicant listed for this patent is CARNEGIE MELLON UNIVERSITY. Invention is credited to Bruce A. Armitage, Saadyah Averick, Subha Ranjan Das, Munira F. Fouz, Krzysztof Matyjaszewski.
Application Number | 20180142288 15/802066 |
Document ID | / |
Family ID | 62144304 |
Filed Date | 2018-05-24 |
United States Patent
Application |
20180142288 |
Kind Code |
A1 |
Fouz; Munira F. ; et
al. |
May 24, 2018 |
NUCLEIC ACID-POLYMER CONJUGATES FOR BRIGHT FLUORESCENT TAGS
Abstract
A composition includes a polymer including extending chains,
side chains, or branches. One (or more) of a plurality of a first
strand of nucleic acid is attached to each of a plurality of the
side chains. One (or more) of a plurality of a second strand of
nucleic acid, which is complementary to the first strand of nucleic
acid, is complexed to each of the plurality of the first strand of
nucleic acid to form a double strand of nucleic acid on each of the
plurality of the side chains. At least one fluorescent compound is
associated with the double strand of nucleic acid on each of the
plurality of the side chains.
Inventors: |
Fouz; Munira F.;
(Gaithersburg, MD) ; Matyjaszewski; Krzysztof;
(Pittsburgh, PA) ; Armitage; Bruce A.;
(Pittsburgh, PA) ; Das; Subha Ranjan; (Pittsburgh,
PA) ; Averick; Saadyah; (Pittsburgh, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CARNEGIE MELLON UNIVERSITY |
Pittsburgh |
PA |
US |
|
|
Family ID: |
62144304 |
Appl. No.: |
15/802066 |
Filed: |
November 2, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62496954 |
Nov 3, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H 21/04 20130101;
C08L 2205/14 20130101; C08L 2203/02 20130101; C08L 57/12 20130101;
C08F 2438/01 20130101; G01N 33/582 20130101; C12Q 1/6834 20130101;
A61K 47/6883 20170801; C08L 57/00 20130101 |
International
Class: |
C12Q 1/6834 20060101
C12Q001/6834; C07H 21/04 20060101 C07H021/04; C08L 57/00 20060101
C08L057/00 |
Goverment Interests
GOVERNMENTAL INTEREST
[0002] This invention was made with government support under
National Science Foundation Award No. 1501324. The government has
certain rights in this invention.
Claims
1. A composition comprising: a polymer comprising side chains, one
of a plurality of a first strand of nucleic acid being attached to
each of a plurality of the side chains, one of a plurality of a
second strand of nucleic acid which is complementary to the first
strand of nucleic acid being complexed to each of the plurality of
the first strand of nucleic acid to form a double strand of nucleic
acid on each of the plurality of the side chains, at least one
fluorescent compound being associated with the double strand of
nucleic acid on each of the plurality of the side chains.
2. The composition of claim 1 further comprising a moiety which has
a binding affinity for one or more target species attached to one
of the side chains.
3. The composition of claim 2 wherein the one of the side chains to
which the moiety is attached comprises a third strand of nucleic
acid, the moiety being attached to a fourth strand of nucleic acid
which is complementary to the third strand of nucleic acid, the
fourth strand of nucleic acid being complexed to the third strand
of nucleic acid.
4. The composition of claim 3 wherein the moiety is an
antibody.
5. The composition of claim 2 wherein the polymer is a bottlebrush
copolymer comprising a backbone from which the side chains extend,
the side chains being formed via a reversible-deactivation radical
polymerization.
6. The composition of claim 5 wherein the at least one fluorescent
compound is associated with the double strand of nucleic acid via
intercalation with the double strands of nucleic acid.
7. The composition of claim 6 wherein a plurality of fluorescent
compounds are intercalated with the double strands of nucleic
acid.
8. The composition of claim 7 wherein each of the double strands of
nucleic acid further comprises at least one fluorescent compound
different from the plurality of fluorescent compounds to which
excitation energy can be transferred to effect a red shift in an
emission wavelength.
9. The composition of claim 8 wherein at least one fluorescent
compound different from the plurality of fluorescent compounds is
covalently attached to the second strand of nucleic acid.
10. The composition of claim 5 wherein the reversible-deactivation
radical polymerization is atom transfer radical polymerization.
11. The composition of claim 2 wherein the at least one fluorescent
compound is covalently attached to the second strand of nucleic
acid.
12. The composition of claim 2 wherein a plurality of fluorescent
compounds are attached to the second strand of nucleic acid.
13. The composition of claim 11 wherein the second strand of
nucleic acid is complementary to a portion of the first strand of
nucleic acid, and the composition further comprises a fifth strand
of nucleic acid which is complementary to a second portion of the
first strand of nucleic acid, the fifth strand of nucleic acid
having at least one fluorescent compound covalently attached
thereto.
14. The composition of claim 13 wherein a plurality of fluorescent
compounds are attached to at least one of the second strand of
nucleic acid or the fifth strand of nucleic acid.
15. The composition of claim 11 wherein the polymer is a
bottlebrush copolymer comprising a backbone from which the side
chains extend, the side chains being formed via a reversible
deactivation radical polymerization.
16. The composition of claim 7 wherein the first strand of nucleic
acid is a first strand of DNA and the second strand of nucleic acid
is a second strand of DNA.
17. A method of labeling a target species, comprising: attaching a
label to the target species which comprises a composition
comprising a polymer having side chains, one of a plurality of a
first strand of nucleic acid being attached to each of a plurality
of the side chains, one of a plurality of a second strand of
nucleic acid which is complementary to the first strand of nucleic
acid being complexed to each of the plurality of the first strand
of nucleic acid to form a double strand of nucleic acid on each of
the plurality of the side chains, at least one fluorescent compound
being associated with the double strand of nucleic acid on each of
the plurality of the side chains.
18. The method of claim 17 wherein the label further comprises a
moiety which has a binding affinity for the target species attached
to one of the side chains.
19. The method of claim 18 wherein the one of the side chains to
which the moiety is attached comprises a third strand of nucleic
acid, the moiety being attached to a fourth strand of nucleic acid
which is complementary to the third strand of nucleic acid, the
fourth strand of nucleic acid being complexed to the third strand
of nucleic acid.
20. The method of claim 19 wherein the moiety is an antibody.
21. The method of claim 20 wherein the antibody is a secondary
antibody.
22. The method of claim 21 wherein the secondary antibody has
affinity for a primary antibody, and the primary antibody has
affinity for the target species.
23. The method of claim 18 wherein the polymer is a bottlebrush
copolymer comprising a backbone from which the side chains extend,
the side chains being formed via a reversible-deactivation radical
polymerization.
24. The method of claim 23 wherein the at least one fluorescent
compound is associated with the double strand of nucleic acid via
intercalation with the double strands of nucleic acid.
25. The method of claim 24 wherein a plurality of fluorescent
compounds are intercalated with the double strands of nucleic
acid.
26. The method of claim 25 wherein each of the double strands of
nucleic acid further comprises at least one fluorescent compound
different from the plurality of fluorescent compounds to which
excitation energy can be transferred to effect a red shift in an
emission wavelength.
27. The method of claim 27 wherein at least one fluorescent
compound different from the plurality of fluorescent compound is
covalently attached to the second strand of nucleic acid.
28. The method of claim 23 wherein the reversible-deactivation
radical polymerization is atom transfer radical polymerization.
29. The method of claim 18 wherein the at least one fluorescent
compound is covalently attached to the second strand of nucleic
acid.
30. The method of claim 18 wherein a plurality of fluorescent
compounds are attached to the second strand of nucleic acid.
31. The method of claim 29 wherein the second strand of nucleic
acid is complementary to a first portion of the first strand of
nucleic acid, and the composition further comprises a fifth strand
of nucleic acid which is complementary to a second portion of the
first strand of nucleic acid, the fifth strand of nucleic acid
having at least one fluorescent compound covalently attached
thereto.
32. The method of claim 31 wherein a plurality of fluorescent
compounds are attached to at least one of the second strand of
nucleic acid or the fifth strand of nucleic acid.
33. The method of claim 29 wherein the polymer is a bottlebrush
copolymer comprising a backbone from which the side chains extend,
the side chains being formed via a reversible-deactivation radical
polymerization.
34. The method of claim 25 wherein the first strand of nucleic acid
is a strand of first strand of DNA and the second strand of nucleic
acid is a second strand of DNA.
35. A method of forming a mobile fluorescent label, comprising:
forming a polymer scaffold having side chains, attaching one of a
plurality of a first strand of nucleic acid to each of a plurality
of the side chains, complexing one of a plurality of a second
strand of nucleic acid which, is complementary to the first strand
of nucleic acid, to each of the plurality of the first strand of
nucleic acid to form a double strand of nucleic acid on each of the
plurality of the side chains, wherein at least one fluorescent
compound is associated with the double strand of nucleic acid on
each of the plurality of the side chains.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional Patent
Application Ser. No. 62/496,954, filed Nov. 3, 2016, the disclosure
of which is incorporated herein by reference.
BACKGROUND
[0003] The following information is provided to assist the reader
in understanding technologies disclosed below and the environment
in which such technologies may typically be used. The terms used
herein are not intended to be limited to any particular narrow
interpretation unless clearly stated otherwise in this document.
References set forth herein may facilitate understanding of the
technologies or the background thereof. The disclosure of all
references cited herein are incorporated by reference.
[0004] Virtually every imaginable aspect of biological systems has
succumbed to labeling through fluorescent probes that have been
developed over the years. Fluorescent dyes coupled to affinity
binders such as antibodies are common reporters in fluorescence
microscopy, flow cytometry and microplate assays as well as in
protein and nucleic acid blots. Despite the advent of competing
approaches such as recombinant peptide tagging and mass
spectrometry, antibody based detection remains the most broadly
applicable means of localizing and quantitating specific components
in a complex sample. Labeled secondary antibodies make stable and
specific complexes with unlabeled primary antibodies, providing the
foundation for most immunofluorescence protocols.
[0005] The number of target molecules per surface area or volume
unit is a key variable in biological detection applications. To
detect functionally important proteins with a natural low
expression level, there remains a need to enhance the detectable
signal The most straightforward way to enhance fluorescence signals
is to increase the number of fluorophores available for detection.
However increased labeling of proteins can lead to precipitation of
the protein.
[0006] As an alternative, signal amplification methods can be used
to get brighter signals. In catalytic reporter deposition (CARD)
methods, the high turnover rate of enzymes like horseradish
peroxidase and alkaline phosphatase generate high density labeling
of a target protein or nucleic acid in situ. Hence in both
immunohistochemical and immunoassay applications, careful control
of timing is an essential prerequisite to obtain quantitative and
re-producible results.
[0007] To avoid these potential limitations of amplification
methods, an alternative is to increase the number of labels
directly on affinity binders. A typical IgG antibody molecule has
about ninety lysine residues, of which at most thirty can be
modified under forcing conditions. However, maintenance of
functional properties typically requires a degree of labeling of
less than ten dyes per IgG, representing a low fraction of
modification with individual fluorescent dyes. Antibodies labeled
with more than four to six fluorophores per protein can exhibit
reduced specificity and binding affinity. With higher degrees of
substitution, the fluorescence obtained per added fluorophore is
typically much lower than expected or calculated, due to
self-quenching.
[0008] The use of soluble and relatively stable fluorescent
proteins such as the phycobiliproteins, conjugated to anti-bodies
could overcome the limitations arising from the high loading of low
molecular weight dyes. On a molar basis, the fluorescence yield of
a phycobiliprotein is equivalent to at least 30 unquenched
fluorescein or 100 rhodamine molecules at comparable wavelengths.
Further, fluorescent polystyrene microspheres heavily loaded with
fluorescent dyes have been used as immuno-fluorescent reagents to
deliver strong signals.
[0009] However, all current labeling schemes for fluorescent dyes
suffer from one or more significant drawbacks. It remains desirable
to provide straightforward and readily usable compositions, systems
and methods for providing bright signals in fluorescent detection
systems.
SUMMARY
[0010] In one aspect, a composition includes a polymer including
extending chains, side chains, or branches. One (or more) of a
plurality of a first strand of nucleic acid is attached to each of
a plurality of the side chains. One of a plurality of a second
strand of nucleic acid, which is complementary (that is, partially
of full complementary) to the first strand of nucleic acid, is
complexed to each of the plurality of the first strand of nucleic
acid to form a double strand of nucleic acid on each of the
plurality of the side chains. At least one fluorescent compound is
associated with the double strand of nucleic acid on each of the
plurality of the side chains.
[0011] In a number of embodiments, the composition further includes
a moiety which has a binding affinity for one or more target
species attached to one of the side chains. The one of the side
chains to which the moiety is attached may, for example, include a
third strand of nucleic acid. The moiety may, for example, be
attached to a fourth strand of nucleic acid which is complementary
to the third strand of nucleic acid, wherein the fourth strand of
nucleic acid is complexed to the third strand of nucleic acid. The
moiety may also be attached to one of the first strands of nucleic
acid via complexing thereto of a strand of nucleic acid, which is
complementary to the first strand of nucleic acid, and which is
attached to the moiety. Alternatively, the moiety may be covalently
attached to the one of the side chains of the polymer. In a number
of embodiments, the moiety is a protein such as an antibody. The
moiety may, for example, also be a peptide (including a cyclic
peptide), an aptamer (that is, an oligonucleotide or peptide
molecule that binds to a specific target molecule), a glycoside, or
a glycopeptides.
[0012] In a number of embodiments, the polymer is a bottlebrush
copolymer including a backbone from which the side chains extend.
The side chains may, for example, be formed via a
reversible-deactivation radical polymerization such an atom
transfer radical polymerization.
[0013] The at least one fluorescent compound may, for example, be
associated with the double strand of nucleic acid via intercalation
with the double strands of nucleic acid. A plurality of fluorescent
compounds may, for example, be intercalated with the double strands
of nucleic acid. In a number of embodiments, each of the double
strands of nucleic acid further includes at least one fluorescent
compound different from the plurality of fluorescent compounds to
effect energy transfer. In a number of embodiments, excitation
energy can be transferred between different fluorescent compounds
(that is, acceptor/donor combinations) to effect a red shift in an
emission wavelength. In a number of embodiment, the at least one
fluorescent compound different from the plurality of fluorescent
compounds is covalently attached to the second strand of nucleic
acid.
[0014] In a number of embodiments, the at least one fluorescent
compound is covalently attached to the second strand of nucleic
acid. A plurality of fluorescent compounds are attached to the
second strand of nucleic acid. One or more of the same or different
fluorescent compounds may be attached to the first strand of
nucleic acid and/or to the second strand of nucleic acid.
Covalently bound fluorescent compounds may be chosen to effect
energy transfer. In a number of embodiments, the second strand of
nucleic acid is complementary to a portion of the first strand of
nucleic acid, and the composition further includes a fifth strand
of nucleic acid which is complementary to another or second portion
of the first strand of nucleic acid. A plurality of fluorescent
compounds may, for example, be attached to at least one of the
second strand of nucleic acid or the fifth strand of nucleic acid.
The polymer may, for example, be a bottlebrush copolymer including
a backbone from which the side chains extend. As described above,
such side chains may, for example, be formed via a
reversible-deactivation radical polymerization such as atom
transfer radical polymerization.
[0015] In a number of embodiments, the first strand of nucleic acid
is a first strand of DNA and the second strand of nucleic acid is a
second strand of DNA.
[0016] In another aspect, a method of labeling a target species
includes attaching a label to the target species which includes a
composition as described above. In that regard, the composition
includes a polymer having side chains. One (or more) of a plurality
of a first strand of nucleic acid is attached to each of a
plurality of the side chains. One of a plurality of a second strand
of nucleic acid, which is complementary to the first strand of
nucleic acid, is complexed to each of the plurality of the first
strand of nucleic acid to form a double strand of nucleic acid on
each of the plurality of the side chains. At least one fluorescent
compound is associated with the double strand of nucleic acid on
each of the plurality of the side chains.
[0017] As described above, the composition may further include a
moiety which has a binding affinity for one or more target species
attached to one of the side chains. The one of the side chains to
which the moiety is attached may, for example, include a third
strand of nucleic acid. The moiety may, for example, be attached to
a fourth strand of nucleic acid which is complementary to the third
strand of nucleic acid, wherein the fourth strand of nucleic acid
is complexed to the third strand of nucleic acid. The moiety may
also be attached to one of the first strands of nucleic acid via
complexing thereto of a strand of nucleic acid, which is
complementary to the first strand of nucleic acid, and which is
attached to the moiety. Alternatively, the moiety may be covalently
attached to the one of the side chains of the polymer. In a number
of embodiments, the moiety is a protein such as an antibody. In a
number of embodiments, the antibody is a secondary antibody. The
secondary antibody may, for example, have affinity for a primary
antibody, and the primary antibody has affinity for the target
species. The label may, for example, include the secondary antibody
and the primary antibody. The moiety may, for example, also be a
peptide (including a cyclic peptide), an aptamer (that is, an
oligonucleotide or peptide molecule that binds to a specific target
molecule), a glycoside, or a glycopeptides.
[0018] As also described above, the polymer may, for example, be a
bottlebrush copolymer including a backbone from which the side
chains extend. The side chains may, for example, be formed via a
reversible-deactivation radical polymerization such an atom
transfer radical polymerization.
[0019] The at least one fluorescent compound may, for example, be
associated with the double strand of nucleic acid via intercalation
with the double strands of nucleic acid. A plurality of fluorescent
compounds may, for example, be intercalated with the double strands
of nucleic acid. In a number of embodiments, each of the double
strands of nucleic acid further includes at least one fluorescent
compound different from the plurality of fluorescent compounds to
effect energy transfer. In a number of embodiments, excitation
energy can be transferred between different fluorescent compounds
(that is, acceptor/donor combinations) to effect a red shift in an
emission wavelength. In a number of embodiment, the at least one
fluorescent compound different from the plurality of fluorescent
compounds is covalently attached to the second strand of nucleic
acid.
[0020] In a number of embodiments, the at least one fluorescent
compound is covalently attached to the second strand of nucleic
acid. A plurality of fluorescent compounds are attached to the
second strand of nucleic acid. One or more of the same or different
fluorescent compounds may be attached to the first strand of
nucleic acid and/or to the second strand of nucleic acid.
Covalently bound fluorescent compounds may be chosen to effect
energy transfer. In a number of embodiments, the second strand of
nucleic acid is complementary to a portion of the first strand of
nucleic acid, and the composition further includes a fifth strand
of nucleic acid which is complementary to another or second portion
of the first strand of nucleic acid. A plurality of fluorescent
compounds may, for example, be attached to at least one of the
second strand of nucleic acid or the fifth strand of nucleic acid.
The polymer may, for example, be a bottlebrush copolymer including
a backbone from which the side chains extend. As described above,
such side chains may, for example, be formed via a
reversible-deactivation radical polymerization such as atom
transfer radical polymerization.
[0021] In a number of embodiments, the first strand of nucleic acid
is a first strand of DNA and the second strand of nucleic acid is a
second strand of DNA.
[0022] In a further aspect, a method of forming a mobile
fluorescent label include forming a polymer scaffold having side
chains, attaching one of a plurality of a first strand of nucleic
acid to each of a plurality of the side chains, complexing one of a
plurality of a second strand of nucleic acid which, is
complementary to the first strand of nucleic acid, to each of the
plurality of the first strand of nucleic acid to form a double
strand of nucleic acid on each of the plurality of the side chains,
wherein at least one fluorescent compound is associated with the
double strand of nucleic acid on each of the plurality of the side
chains. The mobile fluorescent label may be further characterized
as described above.
[0023] The present systems, methods and compositions, along with
the attributes and attendant advantages thereof, will best be
appreciated and understood in view of the following detailed
description taken in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1A illustrates schematically the assembly of a
representative bottlebrush polymer (BBP) with azide side chain
terminii by CuAAC or click reaction with hexynyl-DNA (red strand),
resulting in single stranded DNA functionalized BBP (or DBBP),
wherein annealing of complementary strands (Acomp) provide hundreds
of base pairs for the intercalation of the fluorescent dye YOYO-1
(yellow) in double stranded DBBP, and wherein the inset shows the
structure of the BBP-azide.
[0025] FIG. 1B illustrates synthesis of a representative
bottlebrush polymer of BBP used in the studies hereof
[0026] FIG. 1C illustrates the chemical structure of YOYO-1.
[0027] FIG. 1D illustrates a graph of increasing fluorescence
intensities (.lamda.ex=488 nm) with increasing YOYO-1 up to 5
equivalents relative to DNA.
[0028] FIG. 1E illustrates a graph of the fluorescence emission at
510 nm versus YOYO-1 equivalents relative to DNA concentration and
shows the saturation of the DBBP with YOYO-1 occurs around 5
equivalents as expected.
[0029] FIG. 1F illustrates representative synthetic DNA sequences
and linking groups used in representative studies hereof
[0030] FIG. 2A illustrates schematically wavelength shifting via
Energy Transfer (ET) from intercalated YOYO-1 to terminal Cy5 dyes
(terminal spheres) on each complementary strand of DNA, which
provides for emission at a higher wavelength.
[0031] FIG. 2B illustrates a fluorescence intensity spectra from
DNA attached to a bottlebrush polymer hereof (Brush DNA) wherein
the traces correspond to signals from brush DNAs that include
(+Cy5) or exclude (-Cy5) covalently attached terminal Cy5 on all
the hybridized DNA strands.
[0032] FIG. 2C illustrates a fluorescence intensity spectra for DNA
in solution (Non-brush DNA) wherein the traces correspond to
signals from the Non-brush DNAs that include (+Cy5) or exclude
(-Cy5) covalently attached terminal Cy5.
[0033] FIG. 2D illustrates schematically the manner in which brush
DNA assembles dye molecules at a high local density in a network of
adjacently located YOYO-1 dyes, wherein the YOYO-1 dyes located in
x, y and z directions of the brush-DNA scaffold can contribute
cooperatively to ET by both intra- and inter-duplex mechanisms.
[0034] FIG. 2E illustrates a fluorescence intensity spectra for
Brush DNA wherein the box highlights the higher ET in the brush DNA
as the fluorescence emission is greater when excitation is at 450
nm through the YOYO-1 than when Cy5 is directly excited at 633
nm.
[0035] FIG. 2F illustrates a fluorescence intensity spectra for
Non-brush DNA wherein the box highlights that no difference is
observed when excitation is at 450 nm or excitation is at 633
nm.
[0036] FIG. 3A illustrates a fluorescence intensity spectra for
Non-brush DNA (that is, free DNA in solution) before (-CT) and
after (+CT) addition of calf thymus (CT) DNA in four times excess
of A/Cy5-Acomp[YOYO-1].
[0037] FIG. 3B illustrates a fluorescence intensity spectra for
Brush DNA before (-CT) and after (+CT) addition of calf thymus (CT)
DNA in four times excess of A/Cy5-Acomp[YOYO-1].
[0038] FIG. 3C illustrates the structure of PEO-alkyne and
synthesis of DNA bottlebrush polymers with lower loading of
DNA.
[0039] FIG. 3D illustrates a fluorescence intensity spectra for
Brush DNA before (-CT) and after (+CT) addition of calf thymus (CT)
DNA in four times excess of A/Cy5-Acomp[YOYO-1] for a brush with
lower (that is, 40% lower) DNA loading and including polyethylene
oxide (PEO) chains.
[0040] FIG. 4A illustrates the preparation of a DNA bottlebrush
polymer hereof with a strand of DNA (sequence B) different from
strand A for complexing an affinity binder (for example, an
antibody) or other targeting moiety having a strand of DNA
complementary to the different strand of DNA.
[0041] FIG. 4B illustrates schematically the preparation of a
nano-label or nanotag hereof based upon tethering an antibody to
the DBBP of FIG. 4A, wherein the DBBP can be hybridized to
Cy5-Acomp (via attached sequence A) and to an antibody bearing a
fully complementary B'comp strand (via attached sequence B) and,
subsequently, loaded with YOYO-1, and wherein the single sequence B
per bottlebrush polymer was used to ensure attachment of one
antibody per brush.
[0042] FIG. 4C illustrates schematically a representative
embodiment of a c-myc protein detection system using primary and
secondary antibody interactions, wherein the nanotag or label (X)
is tethered to the secondary antibody.
[0043] FIG. 4D illustrates a UV traceable bisaryl haydrazone
linkage between an antibody and a DNA nucleotide used for
conjugation of the antibody to the DNA bottlebrush polymers
hereof.
[0044] FIG. 4E illustrates flow cytometry data showing high
fluorescent brightness of a representative brush nanotag hereof
compared to commercially available antibody tags.
[0045] FIG. 4F illustrates confocal microscopic images showing the
increased brightness of representative nanotag antibodies hereof
compared to commercially available ALEXA FLUOR 647.RTM.-tagged (a
fluorescent tag available from Thermo Fisher Scientific of Waltham,
Mass.) and QD655-tagged (a fluorescent tag available from Thermo
Fisher Scientific) antibodies
[0046] FIG. 4G illustrates the detection of maltose binding protein
(MBP) using representative primary and secondary antibody system
hereof and the visual confirmation of the specificity and
brightness of DBBP nanotags via dot blots, wherein the nanotag
antibodies hereof allow visualization with at least an order of
magnitude greater sensitivity compared to commercially available
systems.
[0047] FIG. 5A illustrates functionalization of antibody with
succinimidyl-6-hydrazinonicotinate acetone hydrazine (SANH).
[0048] FIG. 5B illustrates functionalization of NH2-B' with
succimidyl-4-formylbenzamide (SFB).
[0049] FIG. 5C illustrates the formation of UV traceable bisaryl
hydrazone linkage between antibody and B'.
[0050] FIG. 6 illustrates a calibration plot of A.sub.260
nm/A.sub.280 nm vs. antibody-DNA ratio to calculate the number of
DNA strands attached per antibody.
[0051] FIG. 7 illustrates schematically several alternative
embodiments of fluorescent tags or labels hereof including
double-stranded DNA or dnDNA attached to the side chain or branch
of a polymer.
DETAILED DESCRIPTION
[0052] It will be readily understood that the components of the
embodiments, as generally described and illustrated in the figures
herein, may be arranged and designed in a wide variety of different
configurations in addition to the described representative
embodiments. Thus, the following more detailed description of the
representative embodiments, as illustrated in the figures, is not
intended to limit the scope of the embodiments, as claimed, but is
merely illustrative of representative embodiments.
[0053] Reference throughout this specification to "one embodiment"
or "an embodiment" (or the like) means that a particular feature,
structure, or characteristic described in connection with the
embodiment is included in at least one embodiment. Thus, the
appearance of the phrases "in one embodiment" or "in an embodiment"
or the like in various places throughout this specification are not
necessarily all referring to the same embodiment.
[0054] Furthermore, described features, structures, or
characteristics may be combined in any suitable manner in one or
more embodiments. In the following description, numerous specific
details are provided to give a thorough understanding of
embodiments. One skilled in the relevant art will recognize,
however, that the various embodiments can be practiced without one
or more of the specific details, or with other methods, components,
materials, et cetera. In other instances, well known structures,
materials, or operations are not shown or described in detail to
avoid obfuscation.
[0055] As used herein and in the appended claims, the singular
forms "a," "an", and "the" include plural references unless the
context clearly dictates otherwise. Thus, for example, reference to
"a fluorescent dye molecule" includes a plurality of such
fluorescent dyes molecules and equivalents thereof known to those
skilled in the art, and so forth, and reference to "the fluorescent
dye molecule" is a reference to one or more such fluorescent dyes
molecules and equivalents thereof known to those skilled in the
art, and so forth. Recitation of ranges of values herein are merely
intended to serve as a shorthand method of referring individually
to each separate value falling within the range. Unless otherwise
indicated herein, and each separate value, as well as intermediate
ranges, are incorporated into the specification as if individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contraindicated by the text.
[0056] The term "polymer" refers generally to a molecule which may
be of high relative molecular mass/weight, the structure of which
includes repeat units derived, actually or conceptually, from
molecules of low relative molecular mass (monomers). The term
"copolymer" refers to a polymer including two or more dissimilar
repeat units (including terpolymers--comprising three dissimilar
repeat units--etc.). The term "oligomer" refers generally to a
molecule of intermediate relative molecular mass, the structure of
which includes a small plurality of units derived, actually or
conceptually, from molecules of lower relative molecular mass
(monomers). In general, a polymer is a compound having >1, and
more typically >10 repeat units or monomer units, while an
oligomer is a compound having >1 and <20, and more typically
leas than ten repeat units or monomer units.
[0057] In a number of embodiments hereof, polymers having multiple
extending chains, side chains or branches are used to attach double
stranded or duplex nucleic acids thereto. In a number of
embodiments, one strand of nucleic acid (a nucleotide or
oligonucleotide) is covalently attached to the side chain,
typically at a terminus thereof. More than one strand of nucleic
acid may be attached to a chain. For example, a doubler or trebler
linking moiety may be used to attach two or three, respectively,
strands of nucleic acid to a chain. As second strand of nucleic
acid, complementary to the first strand may then complexed to the
first strand. In a number of embodiments hereof, the resultant
duplexed nucleic acids are used to associate one or more
fluorescent dye molecules (by intercalation and/or by covalent
bonding) with the polymer-nucleic acid complexes hereof in forming
fluorescent tags or labels. Increasing the number of fluorescent
dye molecules associated with a label or tag hereof can increase
the brightness of the label or tag. In the case that fluorescent
molecules are to be intercalated, forming a duplex is required.
However, in the case that fluorescent molecules/moieties are to be
associated with the extending chains hereof only by covalent
attachment, a single strand of nucleic acid or a duplex may be
present on the extending chains.
[0058] The polymeric labels or tags hereof, when associated with an
affinity binding or other targeting moiety (for example, via
covalent bonding or via nucleic acid hybridization/complexing), are
mobile within a liquid matrix (for example, in solution) or, for
example, on membrane in a blot test. Example of affinity binding
moieties for user herein include proteins (for example,
antibodies), peptides (including cyclic peptides), aptamers
(oligonucleotide or peptide molecules that bind to a specific
target molecule), glycosides, and glycopeptides. As clear to those
skilled in the art, the polymers of the compositions hereof, as
well as the compositions, are soluble or dispersible in aqueous
environments. When, for example, employed with a targeting moiety
or probe which has a binding affinity for one or more target
species, the conjugate composition is mobile in an aqueous sample
to reach and interact with the target species. The polymers hereof
may also be biocompatible. As used herein, the term "biocompatible"
refers to polymers that are compatible with living cells, tissues,
organs or systems, (that is, which do not produce substantial
adverse effect--for example, by inducing excessive inflammation,
excessive cytotoxicity or other excessive response in a living
system), The polymers hereof can range broadly in the number and
molecular weight of side chains or branches. Polymers hereof may,
for example, include one hundred or more extending chains or
branches. In general, the greater the number of side chains (that
is, multiple extending chains), the greater the number of
fluorescent dye molecules that may be associated with the label or
tag, and the brighter the signal output. Polymers having multiple
side chains or branches suitable for user herein include, but are
not limited to, star-shaped polymers, comb polymers, dendrimers,
graft polymers and brush or bottlebrush polymers. Star-shaped
polymer or star polymers are branched or hyperbranched polymers in
which a single branch point or a relatively short backbone chain
gives rise to multiple linear side chains. Comb polymers include a
main chain or backbone with two or more three-way branch points and
linear side chains. A dendrimer is a repetitively branched polymer.
Graft polymers/copolymers have one or more side chains that are
different, structurally or configurationally, from the main chain
or backbone of the polymer. Graft copolymers belong to the general
class of segmented copolymers and generally include a linear
backbone of one composition and randomly distributed branches of a
different composition. The major difference between graft
copolymers and bottlebrush polymers is the grafting density.
Bottlebrush polymer have sufficient grafting density that the
conformational and physical properties of brush polymers are
affected by steric repulsion of the densely grafted side chains.
High graft density along the backbone results is a significant
degree of side chain/side chain interactions, particularly close to
the polymer backbone, resulting in chain extension of the polymer
backbone. Bottlebrush polymers may, for In general, polymer
properties (including, polymer composition, size etc.) can be
readily controlled in the labels or tags hereof for a particular
application. Typically, in a bottlebrush polymer, the polymer
backbone is longer than the chains extending therefrom. If the
chains extending from a polymer backbone are longer than the
backbone the polymer may be referenced as a star polymer.
[0059] The polymers hereof may, for example, be formed via a
grafting from approach, in which the polymer backbone includes
active sites capable of initiating functionality, or a grafting
onto approach, in which the polymer backbone includes functional
groups that are reactive with functional groups of a polymer or
oligomer chain to be grafted onto the backbone. In either grafting
from or grating onto approaches, Reversible-Deactivation Radical
Polymerization (RDRP) procedures, as further described below, may
be used to form the extending chains or branches.
[0060] Although, bright signal outputs are desirable for
fluorescence detection of biomolecules at their native expression
levels, simply increasing the number of labels on a probe,
targeting species or targeting moiety often results in
crowding-induced self-quenching of chromophores. Also, maintaining
the function of the targeting species or moiety (for example, an
antibody) is a concern when increasing the number of labels
thereon. Representative embodiments hereof provide a simple method
to accommodate hundreds or thousands of fluorescent dye molecules
on a single probe, targeting species or targeting moiety (for
example, an affinity binding probe such as an antibody) while
avoiding the negative effects of self-quenching. In a number of
embodiments, polymers including multiple side chains or branching
such as bottlebrush polymer from which extend multiple (for
example, hundreds) of duplex nucleic acid (for example, DNA)
strands are used to accommodate, for example, hundreds of
covalently attached and/or thousands of noncovalently intercalated
fluorescent dye molecules. The polymer-nucleic acid assemblies or
conjugates hereof are bright, prevent dissociation of the
fluorophores, and can be tethered through, for example, nucleic
acid hybridization to a probe (such as an IgG antibody) to make a
bright fluorescent nanotag. Such an antibody (or other affinity
binding entity) fluorescent nanotag may, for example, detect
protein targets in flow cytometry, confocal fluorescence microscopy
and dot blots with an exceptionally bright signal. The signal is at
least an order of magnitude greater than current commercially
available antibodies labeled with organic dyes or quantum dots.
[0061] The branched polymeric scaffolds hereof may thus incorporate
many molecules of one or more fluorescent dyes. In a number of
representative embodiments, the nano-labels or nanotags hereof are
attached to a single antibody to provide a powerful fluorescent
signal that is not achievable with current methods of labeling. In
a number of embodiments, the polymeric scaffold extends hundreds of
double stranded DNA from the side chains of a polymeric core to
assemble intercalating fluorescent dyes. Brush-like polyremes
provide flexible and compact architectures which, as discussed
further below, provide stabilization of intercalated fluorescent
dye molecules. In a number of representative embodiments,
compositions, systems and methods hereof are discussed in
connection with assemblies or conjugates of a bottlebrush polymer
with nucleic acid (DNA) `bristles` grafted to the side chains
thereof. However, one skilled in the art will appreciate that any
polymer including multiple side chains may be conjugated with
nucleic acids for association with chromophore/dye molecules
therewith. Nucleic acids are particularly effective scaffolds for
fluorescent dyes and provide opportunities to design simple
architectures for harnessing and efficiently transporting
energy.
[0062] In a number of embodiments hereof, a Reversible-Deactivation
Radical Polymerization procedure is use to form polymer side chains
in conjugates hereof. Reversible-Deactivation Radical
Polymerization (RDRP) procedures, formerly referred to as
controlled radical polymerization (CRP) procedures, include, for
example, Nitroxide Mediated Polymerization (NMP), Atom Transfer
Radical Polymerization (ATRP), and Reversible Addition
Fragmentation Transfer (RAFT) and others (including cobalt mediated
transfer) that have evolved over the last two decades. RDRP provide
access to polymer and copolymers comprising radically
polymerizable/copolymerizable monomers with predefined molecular
weights, compositions, architectures and narrow/controlled
molecular weight distributions. Because RDRP processes can provide
compositionally homogeneous well-defined polymers, with predicted
molecular weight, narrow/designed molecular weight distribution,
and high degrees of .alpha.- and .omega.-chain
end-functionalization, they have been the subject of much study, as
reported in several review articles and ACS symposia. See, for
example, Qiu, J.; Charleux, B.; Matyjaszewski, K., Prog. Polym.
Sci. 2001, 26, 2083; Davis, K. A.; Matyjaszewski, K. Adv. Polym.
Sci. 2002, 159, 1; Matyjaszewski, K., Ed. Controlled Radical
Polymerization; ACS: Washington, D.C., 1998; ACS Symposium Series
685. Matyjaszewski, K., Ed.; Controlled/Living Radical
Polymerization. Progress in ATRP, NMP, and RAFT; ACS: Washington,
D.C., 2000; ACS Symposium Series 768; and Matyjaszewski, K., Davis,
T. P., Eds. Handbook of Radical Polymerization; Wiley: Hoboken,
2002, the disclosures of which are incorporated herein by
reference.
[0063] A growing number of researchers have used RDRP procedures to
prepare polymeric assemblies by grafting from surfaces or molecules
functionalized with agents to control the selected RDRP procedure,
particularly if it is of importance to control grafting density,
molecular weight and composition of the tethered polymer/copolymer
chain. Matyjaszewski and coworkers disclosed the fundamental four
component ATRP process, comprising the addition, or in situ
formation, of an initiator, in this case a molecule with a
transferable atom or group that is completely incorporated into the
final product, a transition metal and a ligand that forms, at least
a partially soluble transition metal complex that participates in a
reversible redox reaction with the added initiator or a dormant
polymer to form the active species to copolymerize radically
polymerizable monomers, in 1995.
[0064] The basic ATRP process and a number of improvements to the
basic ATRP process have been described in a number of commonly
assigned patents and patent applications including, for example,
U.S. Pat. Nos. 5,763,546; 5,807,937; 5,789,487; 5,945,491;
6,111,022; 6,121,371; 6,124,411; 6,162,882; 6,624,262; 6,407,187;
6,512,060; 6,538,091; 6,541,580; 6,624,262; 6,627,314; 6,759,491;
6,790,919; 6,887,962; 7,019,082; 7,049,373; 7,064,166; 7,125,938;
7,157,530; 7.332,550; 7,407,995; 7,572,874; 7,678,869; 7,795,355;
7,825,199; 7,893,173; 7,893,174; 8,252,880; 8,273,823; 8,349,410;
8,367,051; 8,404,788; 8,445,610; 8,816,001; 8.865,795; 8,871,831;
8,962,764; 9,243,274; 9,410,020; Ser. Nos. 13/99,3521; 14/239,181;
14/379,418; the disclosures of which are incorporated herein by
reference.
[0065] ATRP has also been discussed in numerous publications with
Matyjaszewski as co-author and reviewed in several book chapters.
See, for example, Matyjaszewski, K. et al. ACS Symp. Ser. 1998,
685, 258-283; ACS Symp. Ser. 1998, 713, 96-112; ACS Symp. Ser.
2000, 729, 270-283; ACS Symp. Ser. 2000, 765, 52-71; ACS Symp. Ser.
2000, 768, 2-26; ACS Symposium Series 2003, 854, 2-9; ACS Symp.
Ser. 2009, 1023, 3-13; ACS Symp. Ser. 2012, 1100, 1, and Chem. Rev.
2001, 101, 2921-2990, the disclosures of which are incorporated
herein by reference. These publications, for example, provide
information on the range of suitable transition metals that can
participate in the redox reaction and suitable ligands for the
different transition metals to form transition metal complexes of
differing activities suitable for polymerizing broad range of
exemplified polymerizable (co)monomers in various solvents and
under different activation procedures. See also J. Am. Chem. Soc.,
2014, 136, 6513-6533; and Green Chemistry 2014, 16, 1673, the
disclosures of which are incorporated herein by reference.
[0066] ATRP is the most efficient RDRP method for the preparation
of pure segmented copolymers, since, generally, unlike RAFT it does
not require addition of a standard free radical initiator to
continuously form new polymer chains that do not contain the
desired .alpha.-functional group in a blocking from or a grafting
from reaction thereby producing purer segmented or hybrid products.
In addition, unlike NMP, ATRP does not require high temperatures to
generate the active species by homolytic cleavage of the dormant
chain end, which precludes direct formation of bioconjugates, in
addition to possessing the capacity to copolymerize a much broader
range of radically copolymerizable monomers than NMP.
[0067] ATRP allows the synthesis of .alpha., .omega.-homo and
hetero-telechelic multi-segmented copolymers with a predetermined
degree of polymerization, narrow molecular weight distribution (low
M.sub.w/M.sub.n), incorporating a wide range of functional monomers
and displaying controllable macromolecular structures under mild
reaction conditions. ATRP generally requires addition or formation
of an alkyl halide or (pseudo)halide as an initiator (R--X) or
dormant polymer chain end (P.sub.n--X), and a partially soluble
transition metal complex (Cu, Fe or Ru, for example) capable of
undergoing a one electron redox reaction as a catalyst (although
metal free ATRP procedures have recently been developed). See, for
example, ACS Macro Letters 2015, 4, 192-196, the disclosure of
which is incorporated herein by reference. The generally accepted
mechanism of an ATRP reaction is shown below
##STR00001##
[0068] The methods of atom transfer radical polymerization (ATRP)
have thus yielded rich and diverse polymer architectures. In a
number of embodiments, extending chains, side chains or branches of
polymers hereof may, for example, be grown/grafted from initiators
or from sites of a transfer agent under aqueous conditions or in
the presence of a polar solvent. The extending polymer chains may,
for example, be hydrophilic or water soluble. In representative
embodiments, the monomers for copolymerization in a grafting from
approach may, for example, include an oligo(ethylene oxide)
methacrylate (OEOMA). In general OEOMA monomers are available in
higher purity than other commercially available biocompatible
oligo(ethylene oxide) methacrylates. Many other monomer are also
suitable for user herein such as Di(ethylene glycol) methyl ether
methacrylate (MEO.sub.2MA), 2-(Dimethylamino)ethyl methacrylate,
DMAEMA, acrylamide (AAm), N,N-dimethylacrylamide (DMA), and
N-vinylimidazole (VI), N-isopropylacrylamide (NIPAM) and
2-(methylsulfinyl)ethyl acrylate (MSEA). Representative polymers
side chains include, for example, chains of poly(monomethoxy
poly(ethylene glycol)-(meth)acrylate) (PPEG(M)A),
poly(N-isopropylacrylamide) (PNIPAM), and
poly(N,N-dimethylacrylamide) (PDMA).
[0069] In a number of embodiments, the degree of polymerization of
side chain in a star, brush and other graft macromolecule is
sufficiently high to facilitate relatively high yield of, for
example, a click reaction to tether a strand of nucleic acid to the
side chains and so a complementary strand nucleic acid may be
complexed therewith. In a number of representative embodiments, a
degree of polymerization of at least 25, at least 40 or at least 50
is achieved. In a number of embodiment, the backbone length is
equal to or longer than the longest side chain length.
[0070] For a scaffold that could display a large and
functionalizable array, a representative bottlebrush polymer (BBP)
with reactive azide groups at the tips of the bristles was
synthesized as illustrated in FIGS. 1A and 1B. As illustrated in
FIG. 1B, to obtain the BBP core, a
poly[2-(2-bromoisobutyryloxy)ethyl methacrylate] (PBiBEM)
macro-initiator with a degree of polymerization of 400 (m), was
used as the backbone. From the PBiBEM core, approximately half of
the bromoisobutyrate groups initiated the side chains, leading to
grafting of 200 (m'') side chains with
2-(2-(2-methoxyethoxy)ethoxy)ethyl methacrylate (MEO.sub.3MA)
monomeric units. The 50% initiation efficiency was calculated based
on the content of the azide groups, and it can be related to the
large size of the monomer with long PEO substituent. The degree of
polymerization of side chains was calculated based on absolute
molecular weight of the bottlebrush measured by light scattering
(Mn=8.6.times.10.sup.6) yielding n=180. The bromides at the side
chain termini were then substituted with azides to provide the
BBP-N.sub.3. The "clickable" bottlebrush polymers hereof provide a
high density of side chains for subsequent functionalization with a
5'-hexynyl modified DNA sequence (hexynyl-A in FIG. 1A; Sequence ID
no. 1 in the Sequence Listing hereof). These DNA sequences could,
for example, be grafted to the side chain termini via a
Cu(I)-catalyzed azide alkyne cycloaddition (`click`) reaction. The
pseudo-ligandless click conditions may be optimized for nucleic
acid conjugations in a buffered solution (phosphate buffered saline
(PBS), pH 7.5) with acetonitrile as a minor co-solvent. Three hours
of reaction time was sufficient for near quantitative reaction of
the DNA to the BBP with the disappearance of the azide peak at 2110
cm.sup.-1 in aqueous phase FTIR confirming the completion of the
reaction. Simple purification using molecular weight cut-off
filters provided the single stranded conjugate, A-BBP with about
200 DNA strands per brush.
[0071] Many types of click reactions may be used in the
compositions hereof. Examples of suitable click reactions for use
herein include, but are not limited to, Staudinger ligation,
azide-alkyne cycloaddition (either strain promoted or copper(I)
catalyzed), reaction of tetrazine with trans-cyclooctenes,
disulfide linking reactions, thiolene reactions, hydrazine-aldehyde
reactions, hydrazine-ketone reactions, hydroxyl aminealdehyde
reactions, hydroxyl amine-ketone reactions and Diels-Alder
reactions. In such click reactions to attach a first moiety to a
second moiety, one of the functional groups of the click reaction
is on the first moiety and the other of the functional groups of
the click reaction is on the second moiety.
[0072] To determine the number of bristles on the bottlebrush
polymer, it was first assumed that there were 400 azide-terminated
side chains (corresponding to a grafting efficiency or 100%). When
hexynyl-DNA and azide from BBP are reacted in an equimolar ratio
based on this assumption, half of the hexynyl DNA was recovered
from the filtrate during the product isolation from molecular
weight cut off filters (30 kDa). However, when the product (DBBP)
was tested using FTIR spectrometry, the azide peak had completely
disappeared indicating that all the azides in BBP reacted with DNA.
Therefore, it was concluded that there was an average of 200 rather
than 400 azide-terminated side chains (that is, bristles) per
brush.
[0073] Complexing or annealing of a complementary strand or DNA
(Acomp in FIGS. 1A and 1F; Sequence ID No. 3 in the incorporated
Sequence Listing) completes the formation of the full DNA-BBP
(DBBP) scaffold, A/Acomp-BBP. With about 200 double stranded DNAs
(dsDNA) per brush, this DBBP can accommodate >1000 intercalating
fluorescent dyes. For the DNA intercalating fluorescent dye, YOYO-1
was chosen as a representative example, YOYO-1 is a commercially
available dimer of oxazole-yellow (see FIG. 1C) which has a higher
affinity for dsDNA than the monomeric counterpart. By titrating
YOYO-1 into the A/Acomp-BBP scaffold, the fluorescence intensity at
510 nm could be monitored to determine the loading of the scaffold
with dye. As illustrated in FIG. 1D, the fluorescence increases
monotonically with the accommodation of one YOYO-1 bisintercalator
per four DNA base pairs (bp), and begins to approach saturation
with approximately five YOYO-1 dimers per dsDNA (see the arrow in
FIG. 1E). When bound to dsDNA, YOYO-1 has an extinction coefficient
of 98 900 M.sup.-1 cm.sup.-1 at 491 nm. Given that there are, on
average, 200 dsDNAs per brush with 18bp/duplex and 2 overhanging
bases, at the saturation point with five YOYO-1 dimers, the
extinction coefficient of the YOYO-1 loaded DBBP scaffold
(A/Acomp[YOYO-1]-BBP) reaches .about.10.sup.8 M.sup.-1
cm.sup.-1.
[0074] It is often desirable to red shift the emission wavelength
away from the excitation wavelength to minimize the background
autofluorescence, particularly in biological samples. Wavelength
shifting can be achieved by allowing the light-absorbing
chromophores (donors) to transfer the excitation energy to lower
energy acceptor chromophores that can fluoresce at a longer
wavelength. To incorporate a wavelength shifting property to the
DBBP scaffold, the complementary strand was designed with a
terminal acceptor cyanine-5 (Cy5) dye (Cy5-Acomp in FIG. 1F).
[0075] Efficient energy transfer (ET) between YOYO-1 and Cy5 (see
FIG. 2A) has been demonstrated. Such ET is clearly observed in the
fluorescence emission spectrum of the DNA-BBP with the peak maximum
emerging at 665 nm while a drop occurs in the YOYO-1 fluorescence
at 510 nm (see arrows in FIG. 2B). Based on the quenching of YOYO-1
emission in the presence of Cy5, the ET efficiency is estimated to
be 52%. The emission spectrum of a control sample with the same
concentration of the A/Cy5-Acomp dsDNA free in the buffered
solution (i.e., non-brush DNA) shows ET but at a lower efficiency
compared to DBBP (see FIG. 2C). The brush immobilized DNA shows
higher ET compared to non-brush DNA in solution. The traces in
FIGS. 2B and 2C correspond to DNAs that include (+Cy5 in FIG. 2B)
or exclude (-Cy5 in FIG. 2B), respectively, covalent terminal Cy5
on all the hybridized DNA strands. The ET is calculated based on
the drop in fluorescence at 510 nm (cyan arrow). Although the
double-strand DNA (dsDNA) sequences are identical, (and without
limitation to any mechanism) their three-dimensional arrangement
and localization around the polymeric core likely leads to the
enhanced ET efficiency through an interduplex mechanism, which
would not be possible for the dsDNA in dilute solution.
[0076] As described above, the DBBP contains about 200 Cy5 dyes
incorporated through hybridization of a Cy5-labeled strand to the
clicked complement present at the tips of the brush bristles.
Direct excitation of Cy5 in the DBBP versus free DNA duplex
revealed no significant difference in Cy5 fluorescence intensity,
indicating a lack of self-quenching even within the relatively
dense environment of the DBBP (see FIGS. 2E and 2F). Without
limitation to any mechanism, this observation could result from
stacking of Cy5 dyes on the ends of their respective duplexes. In
contrast, when the samples are excited in the YOYO-1 absorption
band, significantly stronger Cy5 emission is observed for the DBBP,
as a result of the more efficient energy transfer in the polymeric
material.
[0077] As illustrated in FIG. 2D, the brush-DNA assembles the dyes
at a high local density in a network of adjacently located YOYO-1
dyes. The YOYO-1 dyes located in x, y and z directions of the
brush-DNA scaffold can contribute cooperatively to ET by both
intra- and inter-duplex mechanisms. In FIGS. 2D and 2E, the boxes
highlight the higher ET in the brush DNA. The fluorescence emission
is greater when excitation is at 450 nm through the YOYO-1 than
when Cy5 is directly excited at 633 nm in brush-DNA (FIG. 2E),
whereas no such difference is observed in non-brush DNA (FIG.
2F).
[0078] While the ease of assembly of intercalating dyes in a DNA
based scaffold is appealing, the dissociation of dyes from the
intercalating sites can lead to reduced brightness and nonspecific
staining. It has been reported that bisintercalating YOYO-1 readily
dissociates from a DNA three-way junction in the presence of excess
calf thymus (CT) DNA. Therefore the stability of the DBBP nanotag
was tested by adding a four-fold excess of CT (in base pairs). In
these experiments, DBBP-bound YOYO-1 can undergo ET to nearby Cy5
dyes, so dissociation of the intercalator with subsequent binding
to CT DNA should result in a decrease in ET. This result was
observed for a control experiment in which YOYO-1 was pre-bound to
a free (i.e. non-brush) Cy5-labeled DNA duplex and then mixed with
excess CT DNA. As illustrated in FIG. 3A, an immediate drop in ET
upon addition of CT DNA was observed, consistent with rapid
dissociation of the intercalator from the free DNA duplex.
[0079] In stark contrast, when CT DNA is added to the YOYO-1 loaded
DBBP, the ET signal remained unchanged, even after 15 hours,
indicating that the polymeric scaffold with dsDNA facilitates
retention of the intercalators (see FIG. 3B). Without limitation to
any mechanism, the strong retention of YOYO-1 by the DBBP may be
attributed to the electrostatic attraction between the
tetracationic intercalating dye and the relatively dense
polyanionic DNA assembly, as opposed to free DNA, where the
negative charge density of the Cy5-labeled duplex is equivalent to
that of the unlabeled CT-DNA competitor. To support this, a
scaffold was designed with a reduced loading of DNA (that is, 40%
less) as illustrated in FIG. 3C. To compensate for the total
packing of strands extending from the polymeric core, poly(ethylene
oxide) (PEO) was also incorporated to react with the remaining
azide groups (60%) of the BBP during the click conjugation reaction
(see FIG. 3C). The completion of the reaction of the azide groups
was verified by aqueous phase FTIR. After pre-loading with YOYO-1
and mixing with CT-DNA, an immediate drop in ET was observed as
illustrated in FIG. 3D. This result is consistent with the
attribution that the greater negative charge density on the fully
dsDNA loaded brush plays a significant role in suppressing the
dissociation of YOYO-1.
[0080] In a number of further studies, dye-loaded DBBPs hereof were
used as fluorescent nanotags or nano-labels for labeling
applications. In representative studies, a DBBP was conjugated to a
secondary IgG antibody for specific targeting as illustrated in
FIGS. 4A through 4D. Functionalization of secondary antibodies is
appealing because a single secondary antibody can be used with many
different primary antibodies generated against diverse antigens. It
has been shown DNA hybridization can be used to generate
protein-polymer hybrids. Furthermore it was demonstrated that an
antibody-DNA nanotag conjugate may be constructed through
hybridization of dye-labeled DNA strands to a secondary antibody
labeled with the complementary DNA strand. This approach was used
so that a DNA strand on the DBBP could be used to hybridize to a
complementary strand covalently conjugated to an antibody. The
antibody was functionalized with an average of one DNA strand
through a bisaryl hydrazone linkage as illustrated in FIG. 4C. The
IgG conjugated 23-mer strand A' (Sequence ID No. 4 in the
incorporated Sequence Listing) is fully complementary in sequence
to strand A on the DBBP, unlike the Cy5-Acomp strands that include
5 unpaired overhanging residues. Thus, the greater stability of the
23-mer was relied on to be able to hybridize the DBBP to the IgG.
To favor hybridization of a single antibody to each brush, a DBBP
was synthesized in which two different DNA sequences were clicked
onto the bristles as illustrated in FIGS. 4A and 4B. Using
proportionally 0.5% of one DNA sequence (sequence B; Sequence ID
No. 2 in the incorporated Sequence Listing)) along with the
previous sequence A during the click reaction gave, on average, a
single copy of sequence B on each brush (as each brush has 200
azides available for functionalization). This separate sequence B
is fully complementary to the antibody (Ab) conjugated DNA sequence
(sequence B, Sequence ID No. 5 in the incorporated Sequence
Listing). The remaining (199) DNA sequences on the brush were
hybridized as previously described to the Cy5 bearing complementary
strand, Cy5-Acomp, to give the final DBBP-nanotag as illustrated in
FIG. 4B. As clear to one skilled in the art, sequence A or another
sequence may be used to complex to an affinity binding moiety
including a complementary strand of nucleic acid. Moreover, an
affinity binding moiety by be covalently attached to the polymer
hereof via coupling/reactive functionalities (using, for example, a
click reaction as described herein).
[0081] DBBP nanotag of FIG. 4B was tested on yeast cells that
expressed on their surface a single chain variable fragment (scFv)
with a c-Myc binding epitope as illustrated in FIG. 4D. After
incubation with a primary antibody specific for the c-Myc epitope,
the cells were stained with the secondary IgG antibody bearing the
fluorescent DBBP for analysis by flow cytometry and microscopy. The
representative DBBP nanotag was tested on yeast cells that
expressed on their surface a single chain variable fragment (scFv)
with a c-Myc binding epitope. After incubation with a primary
antibody specific for the c-Myc epitope, the cells were stained
with the secondary IgG antibody bearing the fluorescent DBBP for
analysis by flow cytometry and microscopy. Commercially available
Alexa 647 and Quantum Dot (QD) 655 tagged goat anti-mouse secondary
IgGs were used as controls and comparison. Based on the histograms
and the mean fluorescence values as illustrated in FIG. 4E, the
DBBP based nanotags are at least an order of magnitude brighter
compared to the QD-655 tagged antibody labels. At the same
secondary antibody concentration as that of the DBBP nanotag (50
nM), the commercially available Alexa 647-conjugated probe gives
only baseline fluorescence intensity. The signal from emission
channel at 695 nm significantly increases when the DBBP scaffold
contains Cy5 dyes. In addition to demonstrating the significantly
higher brightness of DBBP-conjugated antibodies, these results
suggest that although a rather large polymer brush DNA conjugate
(120.times.20 nm) is tethered to the antibody, antigen binding is
not hindered.
[0082] The confocal images shown in FIG. 4F reinforce the flow
cytometry results. Under the conditions of the studies, the Alexa
647-labeled antibody failed to stain the cells, while staining is
evident for the QD 655 and DBBP-tagged antibodies. Similar labeling
experiments were also performed using biotin coated polystyrene
beads by changing to a mouse anti-biotin primary antibody. In both
flow cytometric and confocal imaging experiments, the DBBP nanotags
again were an order of magnitude brighter than the commercially
available probes. The efficient but incomplete ET in the DBBP
allows strong staining to be observed in all three channels. Given
the brightness of the DBBP nanotag, the reliance on ET to shift the
emission away from autofluorescence is likely unnecessary, since
any background signal will be much weaker than the specific
staining resulting from the DBBP. Furthermore, while control over
the extent of functionalization of the antibody was optimized, the
conjugation was not directed to a specific residue on the protein.
With emerging methods for greater control and specific attachment
of DNA and/or other nucleic acids to antibodies, the hybridization
method can be even more powerful for the development of probes.
[0083] The studies hereof were also extended to dot blots, a widely
used format for screening and detection of specific bio-molecules.
This represents a simplification of the northern blot, Southern
blot, or western blot methods. To evaluate the efficiency of the
DBBP nanotag in this method, maltose binding protein (MBP) was
chosen as a target. MBP is part of the maltose/maltodextrin system
of Escherichia coli, which is responsible for the uptake and
efficient catabolism of maltodextrins. An anti-MBP monoclonal
primary antibody sandwiched between the MBP target blotted on a
nitrocellulose paper, with the DBBP nanotag was chosen as the
detection system (see FIG. 4G). Increasing amounts of MBP blotted
on the paper could be detected with the DBBP nanotag, as shown by
increasing density of the spots in FIG. 4E. The paper was excited
at either 473 nm (YOYO-1) or 650 nm (Cy5 or Alexa 647) and the
emission was recorded at 695 nm. At both excitation wavelengths, as
little as 0.5 ng MBP could be detected by eye for the
DBBP-conjugated antibody. The observation of discrete spots for
excitation of YOYO-1 indicates that the dye remains sequestered by
the DNA within the polymer brush, instead of nonspecifically
staining the paper. In separate experiments using an antibody
conjugated to a DNA tetrahedron nanotag, YOYO-1 was not retained by
the antibody, leading to strong nonspecific background fluorescence
from the paper. The control experiment performed under similar
conditions with an Alexa 647-tagged secondary antibody gave a
detection limit of 5 ng of MBP protein. The DBBP nanotag is thus a
versatile and bright multichromophore system for labelling
applications.
[0084] The use of a nucleic acid-(DNA)-conjugated bottle-brush
polymer as a brightly fluorescent nanotag that is useful in
applications for sensitive detection of proteins both in blots and
directly on cell surfaces has been demonstrated herein. In
particular, it has been shown that the polymer brush architecture
provides the means to graft nucleic acid side chains at a high
local density. The conjugation of nucleic acid to the polymeric
scaffold is readily achieved via, for example, the high efficiency
Cu(I) promoted azide-alkyne cycloaddition reaction with a simple
filtration to purify the brush-nucleic acid conjugate. These brush
localized nucleic acid (for example, DNA) bristles provide a dense
scaffold for intercalating fluorescent dyes such as YOYO-1,
generating extinction coefficients as much as .about.10.sup.8
M.sup.-1 cm.sup.-1 per nanotag. Importantly, the intercalated
YOYO-1 dyes are retained on the DBBP scaffold without dissociation,
avoiding complications from background fluorescence. This property
eliminates the need for obligatory covalent conjugation of dyes and
tremendously eases the assembly of thousands of fluorescent dyes
via simple mixing. The representative approach of using DNA
bristles on the polymer brush provides access to scaffolds with the
same donor chromophore but with additional and different terminal
acceptor dyes at the end of the DNA strands. The ability to excite
each fluorescent scaffold at a single wavelength while monitoring
emission in different channels enables multiplex detection schemes
involving antibodies labeled with unique DBBPs. The high degree of
control over the sequences on the brush allows us to tailor
specific sequences for attaching targeting agents through simple
hybridization and without any further modification to the generic
scaffold design. In the representative examples described herein,
conjugation of the large DBBP to a secondary antibody through DNA
hybridization does not hinder the antibody's binding. It was shown
that these versatile DBBP nanotags on antibodies provide probes
that are at least ten times brighter than commercially available QD
or Alexa fluor-tagged IgG antibodies. By lengthening the nucleic
acid sequences (to provide more intercalation sites) or the number
of nucleic acid strands on the brushes, it is possible to increase
the chromophore content of the antibody nanotags even further.
Using other targeting agents such as an internalizing antibody or
an aptamer or using smaller brushes that are approximately
spherical in shape which can enter cells expands the technology to
detect targets in internal cell compartments or for other types of
in vitro detection assays.
[0085] FIG. 7 illustrates schematically several alternative
embodiments of fluorescent tags or labels hereof including
double-stranded nucleic acid such as DNA attached to the side chain
or branch of a polymer. Embodiment (a) of FIG. 7 is a schematic
representation of the DNA bottlebrush polymer 10a discussed above.
Once again, this embodiment includes two nucleic acid strands 20a
and 30a. First strand 20a is covalently attached to each side chain
100a or bristle of, for example, a bottlebrush polymer, and second,
complementary (either partially or fully complementary) strand 30a
is associated or complexed therewith via hybridization. As used
herein, the term "complementary" refers to either partially
complementary or fully complementary strands of nucleic acid which
may be complexed to form a duplex. Intercalating dyes 200a were
bound noncovalently to the double stranded nucleic acid.
Intercalated dye molecules 200a could generate fluorescence and/or
transfer energy to an optional "acceptor" dye 300a attached
covalently to one of strands 20a and 30a.
[0086] As discussed above, and as demonstrated by the covalent
attachment of an acceptor dye such as Cy5 to one of the DNA strands
(as, for example, discussed in connection with FIGS. 2A through
2F), fluorescent dye molecules can be covalently attached to one or
both of the strands of nucleic acid. In the case that one or more
florescent dye molecules are attached covalently to the nucleic
acid duplex, the concern over dissociation of the dye molecules
discussed above in connection with intercalated dye molecules is
eliminated. As described above, brush polymers scaffolds were found
facilitate retention of the intercalated dye molecules with dsDNA.
Thus, brush polymers may be preferred for use with intercalated
dyes. However, in the case of covalently attached dye molecules,
polymers having multiple side chains that are not bottlebrush
polymers may readily be used. Moreover, DNA often more readily
allows intercalation of fluorescent dye molecules than other
nucleic acids. In the case that all fluorescent dye molecule are
covalently attached to the nucleic acid strands, other synthetic
nucleic acids such as peptide nucleic acids (PNAs), gamma PNAs or
locked nucleic acids (LNAs), which may not bind intercalator dyes
with high affinity, may be used. In a number of embodiments, the
length of the nucleic acid strands covalently attached to the
extending side chains polymer scaffolds hereof is at least 6 mers.
In a number of embodiments, the length of the side chains is in the
range of 6 to 50 mers, or in the range of 6 to 40 mers. The length
of the complementary nucleic acid strands complexed with the
nucleic acid strand covalently attached to the polymer scaffolds
hereof may, for example, also be at least 6 mers, in the range of 6
to 50 mers, or in the range of 6 to 40 mers. The two strands of the
duplex need not be of the same length.
[0087] In the case of covalently bound fluorescent dye molecules or
moieties, it is not necessary in some embodiments, to include a
nucleic acid duplex in the compositions hereof. In that regard, one
or more fluorescent dye molecules or moieties may be covalently
bound to single strand of nucleic acid in the compositions hereof.
Grafted polymers hereof, in which the grafting of many side chains
is possible (either via grafting from or grafting onto a polymer
core) may provide advantages in such embodiments. Moreover, the use
of RDRP to synthesize such side groups provide significant control
of polymer properties and/or inter-nucleic acid/nucleotide
interaction. However, associating one or more fluorescent dye
molecules attached to a strand of nucleic acid by complexing that
strand to a complementary strand of nucleic acid attached to a
polymeric scaffold hereof provide significant advantages in
controlling the formation of the composition (including, for
example, control of the association of different fluorescent dye
molecules to the polymer scaffold). The presence of duplex strands
also provided the capability of associating additional fluorescent
dye molecules via intercalation as described above.
[0088] In embodiments (b), (c) and (d) of FIG. 7, the are no
intercalated fluorescent dye molecules. In those embodiments, all
fluorescent dye molecules are incorporated through covalent bonds
to the nucleic acid strand termini and/or to one or more internal
positions thereon. In embodiment (b), a single fluorescent dye
molecule 300b is attached to strand 30b, which is complexed to a
terminus of nucleic acid strand 20b. In embodiment (c), a plurality
of fluorescent dye molecules 300c are attached to strand 30c (which
is complexed to nucleic acid strand 20c) at the terminus of and
internally to strand 30c. In embodiment (d), multiple short strands
30d and 30'd are complexed or hybridized to a single longer strand
20d as another way to introduce multiple dye molecules 300d per
polymer side chain 100d.
Experimental Section
[0089] Synthesis of Azide Functionalized BBP
[Poly(2-(2-Bromoisobutyryloxy)ethyl
Methacrylate)-graft-Poly(2-(2-(2-Methoxyethoxy)ethoxy)ethyl
Methacrylate) (PBiBEM-g-PMEO.sub.3MA-N.sub.3)] A poly
[2-(2-bromoisobutyryloxy)ethyl methacrylate] (PBiBEM)
macroinitiator with a degree of polymerization in the backbone of
400=m (Figure S10) was synthesized by following a previously
reported procedure.sup.1. 2-(2-(2-methoxyethoxy)ethoxy)ethyl
methacrylate) (MEO.sub.3MA), used as monomer, was polymerized from
PBiBEM macroinitiator by ATRP via a grafting-from method using
Cu(I)Br, Cu(II)Br.sub.2, and 4,4'-dinonyl-2,2'-dipyridyl(dNbpy).
The initial ratio of reagents in the grafting-from reaction was
[MEO.sub.3MA]/[BiBEM]/[Cu(I)Br]/[Cu(II)Br.sub.2]/[dNbpy]=500/1.0/0.9/0.1/-
2.0. MEO.sub.3MA (4.0 g, 17 mmol), PBiBEM (0.0096 g, 0.034 mmol of
bromine initiating groups (BiBEM)), Cu(II)Br.sub.2 (0.00077 g,
0.0034 mmol), dNbpy (0.028 g, 0.069 mmol), and 40% (v/v) anisole
were added to a 25 mL Schlenk flask equipped with a stir bar. The
mixture was degassed via three freeze-pump-thaw cycles, and then
Cu(I)Br (0.0044 g, 0.031 mmol) was added when the solution was
frozen during the final cycle. The flask was sealed and placed in
an oil bath at 25.degree. C. Conversion was analyzed by gas
chromatography (GC) using anisole as the internal standard. The
polymerization was stopped after 6 h and the flask was opened to
air. The solution was passed through a column of neutral alumina,
and then precipitated into hexanes three times. Finally the
resulting polymer was dialyzed against three changes of THF for 2
days. The polymer was dried under vacuum at room temperature for 24
h providing PBiBEM400-g-PMEO.sub.3MA180 with a degree of
polymerization in the side chain of MEO.sub.3MA=180 estimated by
GC. A Schlenk flask was charged with PBiBEM400-g-PMEO.sub.3MA180
(1.0 g, 0.088.times.10.sup.-3 mmol), sodium azide (0.023 g, 0.35
mmol), and 10 mL DMF. The reaction mixture was stirred at room
temperature for 48 h. The solution was dialyzed against four
changes of DMF for 3 days to remove excess sodium azide, and then
the DMF solvent in dialysis was replaced with THF. The solution was
evaporated and dried under vacuum at room temperature for 24 h. The
polymer was analyzed by .sup.1H-NMR spectroscopy in CDCl.sub.3, gel
permeation chromatography (GPC) in THF, and FTIR spectroscopy
providing PBiBEM400-g-PMEO.sub.3MA180-N.sub.3:
Mn=8.6.times.10.sup.6, Mw/Mn=1.29, and 2110 cm.sup.-1
(--N.dbd.N+.dbd.N--).
[0090] Oligonucleotides. DNA sequences with 5' terminal hexynyl and
5' Cy5 modifications were synthesized using solid phase
oligonucleotide synthesis on a MerMade-4 synthesizer
(Bio-automation, Plano, Tex., USA). Commercially available starting
materials were used without further purification. Phosphoramidites
(dA, dC, dG and T) with labile PAC protecting groups and
appropriate reagents were purchased from ChemGenes (Wilmington,
Mass. USA) and Glen Research (Sterling, Va. USA). Synthesis and
de-protection of the oligonucleotides were conducted under standard
protocols for PAC-protected amidites, as recommended by the
manufacturers. The DNA synthesis columns were purchased from
Biosearch Technologies, Inc (Novato, Calif. USA). 5'-amino-modified
DNA and any unmodified DNA oligonucleotides were purchased from
Integrated DNA Technologies, Inc. and obtained as lyophilized
powders. The DNA sequences are illustrated in FIG. 1F in which
underlining indicated the complementary regions.
[0091] Click Conjugation of Hexynyl-A to BBP-N.sub.3. BBP-N.sub.3
(4 mg) was dissolved in 100 .mu.L of deionized (18.0 MQ) H.sub.2O
to give a 1 mM azide stock solution. A lyophilized powder of
hexynyl-A was dissolved in H.sub.2O to obtain a 500 .mu.M hexynyl
stock solution. The BBP-N.sub.3 (10 .mu.L, 1 mM azide) and
hexynyl-A (20 .mu.L, 500 .mu.M) solutions were mixed and degassed
several times by blowing with argon, to remove any dissolved
oxygen. Acetonitrile (1.5 .mu.L, 20% ACN in H.sub.2O V/V) and a
freshly prepared solution of sodium ascorbate prepared in deionized
H.sub.2O (8 .mu.L, 100 mM) were mixed and degassed in a separate
vial. The two solutions were added to 4.5 .mu.L of degassed
deionized H.sub.2O and the final pH and salt concentration were
brought to pH=7.5 and 100 mM Na with 5 .mu.L of 10.times. PBS
buffer. The solution was mixed thoroughly and degassed. A degassed
solution of CuSO.sub.4 (1 .mu.L, 100 mM) was added to initiate the
reaction and was allowed to run for 3 hours at room temperature
with gentle shaking. The DBBP product was purified with a 30 kDa
molecular weight cutoff filter (Millipore) and dissolved in PBS
buffer (pH=7.5, =100 mM). The DNA concentration was calculated
using the UV absorbance at 260 nm using a NanoDrop UV/Vis
spectrometer. The final DBBP product was stored in a -14.degree. C.
freezer to be used later. For solution phase FTIR experiments the
isolated DBBP from the molecular weight cutoff filter was dissolved
in deionized water instead of PBS buffer.
[0092] Synthesis of A-BBP with 40% Saturation of Azide Terminating
Side Chains. The general procedure for the conjugation of
hexynyl-DNA to BBP-N.sub.3 described above was followed by using
40% as much hexynyl-A DNA (8 .mu.L, 500 .mu.M stock).
[0093] Synthesis of PEO/A-BBP with 40% Hexynyl-A and 60% PEO-alkyne
by Sequential Click Conjugation. An alkyne-terminated poly(ethylene
oxide)(PEO-alkyne) synthesized using a previously reported
procedure.sup.2 was used for the click reaction. PEO-alkyne (2.08
mg) was dissolved in 5 mL of deionized water to make a 200 .mu.M
stock of alkyne. Azide from BBP-N.sub.3 (10 .mu.L, 1 mM) and
hexynyl-A (8 .mu.L, 500 .mu.M) were mixed and degassed several
times to remove any dissolved oxygen. Acetonitrile (1.5 .mu.L, 20%
ACN in H.sub.2O V/V) and a freshly prepared solution of sodium
ascorbate prepared in deionized H.sub.2O (8 .mu.L, 100 mM) were
mixed and degassed in a separate vial. The two solutions were added
to 1.5 .mu.L of degassed deionized H.sub.2O and the final pH and
salt concentration were brought to pH=7.5 and 100 mM Na.sup.+ with
5 .mu.L of 10.times. PBS buffer. The solution was mixed thoroughly
and degassed. A degassed solution of CuSO.sub.4 (1 .mu.L, 100 mM)
was added to initiate the reaction and was allowed to run for 1
hour at room temperature with gentle shaking. After 1 hour a
degassed solution of PEO-alkyne (15 .mu.L, 200 .mu.M) was added and
the reaction was allowed to run for two more hours. The DBBP
product was purified using molecular weight cutoff filters.
[0094] Synthesis of A/B-BBP with 99.5% Hexynyl-A and 0.5%
Hexynyl-B. The sequential click conjugation method illustrated in
FIG. 4A was followed using BBP-N.sub.3 (10 .mu.L, 1 mM azide),
hexynyl-B (5 .mu.L, 10 .mu.M) and hexynyl-A (19.9 .mu.L, 500
.mu.M).
[0095] Hybridization of DNA Duplexes and YOYO-1 Intercalation. An
equimolar mixture of Seq.A from DBBP and Cy5-Acomp were mixed
together in buffered aqueous solution (PBS buffer, pH=7.5, 100 mM
Nat) to give a 100 nM final concentration of duplex DNA. Nonbrush
DNA duplexes were prepared by mixing seq. A and Cy5-Acomp in an
equimolar ratio in buffered solution to give the same final
concentration of duplex DNA. The same step was followed to prepare
bush and nonbrush duplex DNA without Cy5 dye by using A.sub.comp in
place of Cy5-A.sub.comp. The vials were incubated in 90.degree. C.
water bath for 2 minutes and quickly transferred to a water bath at
65.degree. C. After incubating for 15 minutes at 65.degree. C. the
samples are cooled down to 25.degree. C. within a period of 1 hour.
The DBBP samples with 40% loading of seq. A were mixed with
equimolar ratio of Cy5-A.sub.comp and A.sub.comp separately in
buffered solutions to give double stranded DBBPs with low loading
of DNA.
[0096] Preparation of Antibody-DNA Conjugates. Antibody-DNA
conjugation (see, for example, FIG. 5A-5C) was performed using the
Solulink protein-oligo conjugation kit (catalog S-9011-1, Solulink)
which uses bisaryl hydrazone conjugation chemistry. Antibody and
DNA modification reactions were optimized to obtain a single DNA
strand conjugated per antibody. Prior to functionalization,
affinity-purified, unlabeled goat-antimouse IgG antibody (Jackson
Immunoresearch) was desalted using MicroSpin G-50 columns (GE
Healthcare) and buffer exchanged (modification buffer, 100 mM
phosphate, 150 mM NaCl, pH 8) using molecular weight cutoff filters
(50 kDa, Millipore Corporation) to bring the final protein
concentration to 2.9 mg/mL. 1.1 mg of SANH (succinimidyl
6-hydrazinonicotinamide acetone hydrazine) reagent provided in the
kit was dissolved in 150 .mu.L of N,N-dimethylformamide (DMF). The
SANH solution and the desalted, buffer-exchanged antibody was mixed
in a 5:1 (SANH:antibody) molar ratio. Separately 1.7 mg of
Hydralink SFB (Succinimidyl 4-formylbenzoate) reagent provided in
the kit was dissolved in 80 .mu.L of DMF. 5'-aminated DNA (NH2-B',
Integrated DNA Technologies, Inc.) was dissolved in modification
buffer to make a 1 mM stock solution. The SFB reagent and the
NH.sub.2--B' was mixed in a 5:1 (SFB:DNA) molar ratio. Both
antibody and DNA coupling reactions were performed at room
temperature for 2 hours on a gently rotating shaker. Excess SANH
and SFB were removed using MicroSpin G-50 and G-25 columns,
respectively. The modified antibody and the DNA were dissolved in
conjugation buffer (100 mM phosphate, 150 mM NaCl, pH 6). Molar
substitution ratio (MSR) assays were performed using the standard
protocols provided by the vendor to determine the number of
modifications per antibody and DNA strand. SFB-derivatized DNA was
then combined with the SANH-derivatized antibody in 1:5 ratio and
allowed to react overnight at room temperature on a gently rotating
shaker. Unreacted DNA was removed using 50 kDa filters. Fractions
from each filtration round were tested for presence of unbound free
DNA (A.sub.260 using NanoDrop spectrophotometer). The conjugation
of antibody-DNA was verified spectrophotometrically, by the
presence of the bisaryl hydrazone linkage (.lamda..sub.max=354 nm)
formed between SANH and SFB.
[0097] Determination of Antibody:DNA Ratio. To quantitatively
determine the number of DNA strands conjugated per antibody, a
calibration experiment was conducted. 20 .mu.L solutions with
various ratios of antibody to DNA were mixed as follows: 1:0 (4
.mu.M and 0 .mu.M, respectively), 1:0.5 (4 .mu.M and 2 .mu.M,
respectively), 1:2 (4 and 8 .mu.M, respectively), 1:4 (4 and 16
.mu.M, respectively), and 1:5 (4 and 20 .mu.M, respectively). Using
the NanoDrop spectrophotometer, the A.sub.260 nm/A.sub.280 nm
absorbance ratios were determined and recorded for each solution.
The obtained calibration curve was then used to determine the
number of DNA strands per antibody (FIG. 6). This procedure
accounts for the fact that the absorbance of the DNA (260 nm)
overlaps with the absorbance of the antibody (280 nm). For the
Ab-B' conjugates reported here, an average of one DNA strand was
attached per antibody.
[0098] Antibody-DBBP Hybridization DBBP with Seq. B: Seq A in a
1:199 ratio was first hybridized with Cy5-Acomp by mixing equimolar
amount of Cy5-Acomp and Seq. A on DBBP in PBS buffer (100 mM Nat,
pH 7.5) and annealing. The resulting DBBP should have, on average,
one unhybridized Seq.B strand available for hybridization to the
antibody functionalized with its complement (Ab-B'). Antibody and
DBBP (10 .mu.M each final concentration) were mixed and allowed to
hybridize at 25.degree. C. overnight on a gently rotating shaker.
The antibody-conjugated DBBP (10 .mu.M) was stored at 4.degree. C.
until further use.
[0099] Labeling and Detection of c-myc Targets of Yeast Cells with
Antibody-DBBP Nanotag. Yeast cells expressing c-myc-tagged scFv
(10.sup.7 cells) were suspended in calcium- and magnesium-free PBS
wash buffer (pH=7.5, Na.sup.+=100 mM, 1 .mu.g/mL PLURONIC.RTM.
F-127, a non-ionic surfactant polyol available from Thermo Fisher
Scientific). The cells were incubated with 0.5 .mu.M final
concentration of anti-c-myc mouse antibody for half an hour at
4.degree. C., washed three times with 500 .mu.L of wash buffer, and
resuspended in 500 .mu.L of the same buffer. The cells were
incubated with 50 nM final concentration of goat anti-mouse
antibody-DBBP complex for 30 minutes at 4.degree. C. The cells were
washed three times with 500 .mu.L wash buffer. YOYO-1 was added to
give a 50 .mu.M final concentration in a total volume of 500 .mu.L
PBS buffer (pH=7.5, Na.sup.+=100 mM, 1 .mu.g/mL PLURONIC.RTM.
F-127). Similarly, 50 nM final concentrations of Alexa 647-tagged
goat anti-mouse IgG (H+L) (Thermo Fisher Scientific) and Qdot.RTM.
655 tagged goat anti-mouse IgG (H+L) (Thermo Fisher Scientific)
were separately used as controls. Labeled cells were analyzed by
fluorescence activated cell sorting (FACS) using a Coulter Epix
Elite flow cytometer (Beckman-Coulter, Fullerton, Calif.). The
following dichroic lenses (DL)/band-pass (BP) filters were used:
550DL/530BP for YOYO-1-donor fluorescence channel, and 720DL/695 BP
for Cy5-acceptor fluorescence channel. Further, a Carl Zeiss LSM
510 Meta DuoScan Inverted Spectral Confocal Microscope was used for
fluorescence imaging analysis at 100.times. objective
magnification. Yeast cell samples were prepared according to the
yeast cell fluorescent staining protocol outlined above, except the
final volume of each sample was 60 .mu.L of PBS wash buffer
(pH=7.5, Na.sup.+=100 mM, 1 .mu.g/mL PLURONIC.RTM. F-127). 20 .mu.L
of the cell suspension was placed on a 35 mm glass bottom micro
well dish (Mattek, part no. P35G-1.5-14-C). The micro well plates
for yeast cells were treated with concanavalin A. Enough wash
buffer was added to fill the micro well before viewing under the
microscope. The pin hole opening was set to the minimum setting and
the tube current was set to 0.6 A for imaging. Raw images were
collected using ZEN 2009 software without applying any digital
enhancements.
[0100] Labeling and Detection of Biotin Targets on Polystyrene
Beads by Antibody-DBBP Nanotag. Biotin-coated polystyrene beads
(10.sup.6 beads) were suspended in 60 .mu.L total volume of
calcium- and magnesium-free PBS (pH=7.5, Na.sup.+=100 mM, 0.02%
Triton X-100) buffer. The beads were incubated with 0.5 .mu.M final
concentration of anti-biotin mouse IgG(H+L) (Jackson
Immunoresearch) for half an hour at 25.degree. C., washed three
times with 500 .mu.L of wash buffer, and resuspended in 500 .mu.L
of the same buffer. The beads were incubated with 50 nM final
concentration of goat anti-mouse antibody-DBBP complex for 30
minutes at 25.degree. C. The beads were washed three times with 500
.mu.L wash buffer. YOYO-1 was added to give a 50 .mu.M final
concentration in a total volume of 500 .mu.L PBS buffer (pH=7.5,
Na.sup.+=100 mM, 1 .mu.g/mL Triton X-100). Similarly, 50 nM final
concentrations of Alexa 647-tagged goat anti-mouse IgG (H+L)
(Thermo Fisher Scientific) and Qdot.RTM. 655 tagged goat anti-mouse
IgG (H+L) (Thermo Fisher Scientific) were separately used as
controls. Samples were allowed to incubate for 30 minutes at
25.degree. C. and washed with 150 .mu.L PBS. Samples were
resuspended in 500 .mu.L PBS. Labeled beads were analyzed by
fluorescence activated cell sorting (FACS) using a Coulter Epix
Elite flow cytometer (Beckman-Coulter, Fullerton, Calif.). The
following dichroic lenses (DL)/band-pass (BP) filters were used:
550DL/530BP for YOYO-1 donor fluorescence channel, and 720DL/695 BP
for Cy5 acceptor fluorescence channel. Further, a Carl Zeiss LSM
510 Meta DuoScan Inverted Spectral Confocal Microscope was used for
fluorescence imaging analysis at 100.times. objective
magnification. Bead samples were prepared according to the
microbead fluorescent staining protocol outlined above, except the
final volume of each sample was 60 .mu.L of PBS wash buffer
(pH=7.5, Na.sup.+=100 mM, 1 .mu.g/mL Triton X-100). 20 .mu.L of the
bead suspension was placed on a 35 mm glass bottom microwell dish
(Mattek, part no. P35G-1.5-14-C). Enough wash buffer was added to
fill the microwell before viewing under the microscope. The pin
hole opening was set to the minimum setting and the tube current
was set to 0.6 A for imaging. Raw images were collected using ZEN
2009 software without applying any digital enhancements.
[0101] Dot Blot Experiments. Maltose-binding protein was blotted on
a pure nitrocellulose transfer and immobilization paper (pore size
0.45 .mu.m, Perkin Elmer) in increasing amounts. (spots of 0.5 ng
to 25 ng in duplicate to get two rows) and was allowed to dry for
30 mins at 25.degree. C. Non-specific binding was inhibited with
blocking buffer (5% milk) for 1 hour at 25.degree. C. The 5% milk
was prepared in Tris-buffered saline and Tween (TBST) buffer
(pH=8.5). The membrane was washed three times using the TBST buffer
to get rid of any unbound protein and excess milk. 1 .mu.L of the
anti-MBP monoclonal antibody-HRP conjugate (New England Biolabs)
was added to 1 mL of 5% milk (1:1000 dilution) and the membrane was
allowed to be covered in this solution containing the primary
antibody overnight at 4.degree. C. with gentle shaking. The
membrane was washed in 25 mL TBST for 5 minutes with gentle
shaking, 3 times for 2 minutes each. A 1:1000 dilution was made
with goat anti-mouse IgG-Alexa 647 conjugate or the goat anti-mouse
IgG-DBBP-nanotag with 1 ml TBST to get similar concentrations of
the secondary antibody (final concentration=2 .mu.g/mL=ca. 10 nM).
The membrane was incubated in the solution for 3 hours. The washing
steps were performed in triplicate with TBST and TBS buffers. The
membrane was dried and was imaged using the Typhoon FLA 9000
scanner. YOYO-1 was excited using the excitation wavelength at 473
nm at 250 V and the direct excitation of Cy5 and Alexa 647 was
performed at 650 nm at 250 V. The blots were scanned to get the
emission at 695 nm.
[0102] Fluorescence measurements. Fluorescence measurements were
taken in 0.1 .mu.M final concentrations of duplex DNA. YOYO-1
(available from Thermo Fisher Scientific). YOYO-1 was added in 0.5
.mu.M final concentration to the pre-annealed DNA duplexes and
mixed for one minute before taking fluorescence measurements.
Energy transfer signals were monitored by exciting the donor YOYO-1
at 450 nm and the resulting emission of Cy5 was measured at 670 nm.
The band passes for both excitation and emission monochromators
were 5 nm. The Energy Transfer efficiency (ET) was calculated on
the basis of the fluorescence intensity of the donor in the
presence (FDA) and absence (FD) of the acceptor dye based to the
following equation: ET=1-(F.sub.DA/F.sub.D).
[0103] The foregoing description and accompanying drawings set
forth a number of representative embodiments at the present time.
Various modifications, additions and alternative designs will, of
course, become apparent to those skilled in the art in light of the
foregoing teachings without departing from the scope hereof, which
is indicated by the following claims rather than by the foregoing
description. All changes and variations that fall within the
meaning and range of equivalency of the claims are to be embraced
within their scope.
Sequence CWU 1
1
5123DNAArtificial SequenceSynthetic construct 1gcactgcagt
tggatcccat agc 23223DNAArtificial SequenceSynthetic Construct
2gctatccatc agaattcgcg acg 23323DNAArtificial SequenceSynthetic
Construct 3atcgagctat gggatccaac tgc 23423DNAArtificial
SequenceSynthetic Construct 4gctatgggat ccaactgcag tgc
23528DNAArtificial SequenceSynthetic construct 5atcgacgtcg
cgaattctga tggatagc 28
* * * * *